My major research goal is to understand how the kinetochore functions in yeast. We have constructed and analyzed mutations in centromere DNA and made significant contributions to understanding the roles played by different CEN sequences in mitotic and meiotic chromosome segregation. We have found that CEN mutants which exhibit reduced function also have an altered centromeric chromatin structure. We are now extending this work by analyzing the CEN DNA:protein interactions in more detail using in vivo footprinting. However, these studies provide little information about the nature of the centromeric components. Therefore we have developed a clever genetic screen to isolate mutants defective in centromere- related functions (crfl-4). These mutants exhibit increased nondisjunction of chromosomes containing mutant CEN DNA but show little increase in loss of WT chromosomes. We are very excited that two of the crf mutants exhibit allele specificity, that is, they have different affects on the function of different centromere mutants. This is strong evidence that the crf mutation affects components of the centromere. Analysis of conditional lethal crf mutants is a top priority. This includes cloning the CRF genes, identifying the corresponding gene products, and investigating the role of these proteins in centromere function. My research group has expertise in molecular biology and yeast genetics, but little experience with proteins or antibodies. Due to the current constraints on my time, and in the absence of senior personnel in my lab, progress in these critical areas would ordinarily rely on the efforts of graduate students. The University has agreed that RCDA released funds will be used to hire a Research Associate to do my classroom teaching. This would permit me time to learn necessary new techniques from two local colleagues. The Research Associate will also contribute to research in my lab. How successfully my research moves in new directions will depend, at least in part, on my ability to provide the necessary impetus and guidance. The opportunities provided by an RCDA would ensure that I make the most of the unique advantage afforded by the crf mutants and thereby significantly advance my research standing in a very competitive field.